The purpose of this project is to elucidate the mechanism of action of the ribosome in protein synthesis by examining the functional roles of individual ribosomal protein and RNA components and the relationships among various partial activities of the ribosome. By means of chemical modification of ribosomes from dissociated molecular components, minimal structure alterations will be introduced into the ribosome. The resulting functional lesions will be characterized in detail by means of a variety of partial reaction assays which measure aspects of ribosome activity. The altered molecular components, and in some cases the functional group, responsible for the defect will be identified by constructing ribosomes containing a single modified component. In this way functional roles will be assigned to individual ribosomal proteins and RNA. In addition characterization of a variety of defective ribosomes produced in this way will elucidate the relationships among the partial activities. These experiments will concentrate on the large (50S) ribosomal subunit from Bacillus stearothermophilus. These experiments have identified three Escherichia coli proteins (L2, L4, and L16) as probable candidates for participation in the peptidyl transferase center of the ribosome. More detailed examination of the B. stearothermophilus homolog of L2 (B-L3) has suggested that it is a multifunctional protein with functional groups that are essential for several active sites. Experiments are proposed to examine the function of this protein further. In addition, the B. Stearothermophilus homologs of L4 and L16 (probably B-l4 and B-L20b, respectively) will be subjected to similar analysis.